Method and device for laminar flow on a sensing surface

ABSTRACT

Methods and devices are provided for controlling a fluid flow over a sensing surface within a flow cell. The methods employ laminar flow techniques to position a fluid flow over one or more discrete sensing areas on the sensing surface of the flow cell. Such methods permit selective sensitization of the discrete sensing areas, and provide selective contact of the discrete sensing areas with a sample fluid flow. Immobilization of a ligand upon the discrete sensing area, followed by selective contact with an analyte contained within the sample fluid flow, allows analysis by a wide variety of techniques. Sensitized sensing surfaces, and sensor devices and systems are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.09/760,213, filed Jan. 12, 2001 (now allowed); which is a continuationof U.S. patent application Ser. No. 09/009,139, filed Jan. 20, 1998(U.S. Pat. No. 6,200,814); both of which applications are incorporatedherein by reference in their entireties.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention generally relates to the control of a fluid flowover a sensing surface within a flow cell and, more specifically, to theuse of laminar flow techniques to position a fluid flow over one or morediscrete sensing areas within a flow cell, as well as the use thereof inthe preparation of sensing surfaces and in analytical methods, devicesand systems related thereto.

2. Description of the Related Art

Instrumentation for real-time Biomolecular Interaction Analysis (BIA) iscommercially available from Biacore AB (Uppsala, Sweden) under the tradename BIAcore (hereinafter “the BIAcore instrument”). The BIAcoreinstrument employs surface plasmon resonance (SPR) to investigateinteractions between molecules at the surface of a sensor chip, andincludes a processing unit with liquid handling and optical systems, asensor chip, and a computer for control and data evaluation. Onemolecule, referred to as the “ligand,” is immobilized on the surface ofthe sensor chip, and the other molecule, the “analyte,” flows over thesurface of the sensor chip. As the analyte interacts with theimmobilized ligand, SPR is used to measure a change in refractive indexon the surface of the sensor chip. Selective interactions of the analyteto the immobilized ligand gives this technique specificity, and alsoenables analysis of interactions in complex mixtures.

The BIAcore instrument has been used extensively, and a large volume ofliterature has been published concerning its operation andapplicability. For example, published PCT WO 90/05295 discloses ingreater detail the construction and operation of the BIAcore instrument,while published PCT applications WO 90/05303 and WO 90/05305 aredirected to various sensor surfaces for use therewith. Further, theBIAtechnology, BIAapplication, and BIAcore Handbooks published byBIAcore AB describe in considerable detail the operation andconstruction of the BIAcore instrument.

In general, an analyte present within a liquid sample interacts with theligand associated with, for example, a dextran matrix -bound to thesurface of the sensor chip. Binding of the analyte by the ligand givesrise to an increase in refractive index which is monitored in real timeby a change in the resonance angle as measured by SPR. The data take theform of a sensorgram that plots the signal in resonance units (RU) as afunction of time. A signal of 1,000 RU corresponds to the binding ofabout 1 ng of analyte per mm² (Johnsson et al., Anal. Biochem.198:268-277, 1991; Fagerstam et al., J. Chromatography 597:397-410,1992; Stenberg et al., Colloid and Interface Science 143:513-526, 1991).

During operation of the BIAcore instrument, the sample is delivered tothe sensor chip utilizing an integrated micro-fluidic cartridge (IFC).The IFC consists of a series of precision-cast channels in a hardsilicon polymer plate, forming sample loops and flow channels for bufferand sample delivery. The IFC is pressed into contact with the sensorchip by a docking mechanism within the BIAcore instrument. Arepresentative IFC as used by the BIAcore instrument is depicted in FIG.1A, which illustrates the channels and valves (as viewed from above),with the inset showing a side view of the same and depicting a flow cellformed from pressing the IFC against the sensor chip.

Sample flow through the IFC is controlled by a set of pneumaticallyactuated diaphragm valves that direct the sample through the variouschannels to the sensing surface of the sensor chip. In this manner, theBIAcore instrument (e.g., BIAcore 2000) permits single or multichannelanalysis in up to four flow cells. For example, FIG. 1B illustratessample being passed through three flow cells in series (labeled FC 1, FC2 and FC 3). Although not specifically depicted in FIG. 1B, sample canalso pass through just a single flow cell for analysis (e.g., FC 1).

Existing BIAcore instruments employ flow cells having a cross sectionalarea of 0.05×0.5 mm and a length of 2.4 mm, giving a volume of about 60nanoliters (nl), and having a total sensing surface area in each flowcell of approximately 1.2 mm². A focused incident light illuminatesapproximately 1.6 mm of the length of the sensing surface for each flowcell, with the detector imaging about 0.17 mm of the width of thesensing surface. This corresponds to a sensing area within each flowcell of about 0.3 mm². Each flow cell in the BIAcore instrument containsa single sensing area. Thus, if the sample is to contact four differentsensing areas, passage of the sample through four separate flow cells isrequired (i.e., FC 1, FC 2, FC 3 and FC 4).

While sample delivery to multiple flow cells as presently employed inthe BIAcore instrument offers numerous advantages, and represents thestate of the art with respect to sample delivery techniques,improvements thereto are still desired. For example, in the context ofkinetic measurements, it is important that sample be delivered to eachflow cell in a well-defined volume or “plug,” with minimal dispersion atsample-buffer boundaries. Such a sample plug is created by switchingbetween sample and buffer flow in the IFC with aid of the pneumaticvalves. While dispersion is minimized by keeping dead volumes betweenthe valves and flow cells small, there are still periods at the startand end of sample introduction where the concentration of the sample isdiluted by dispersion (i.e., mixing of the sample with the runningbuffer in the system). Further, dispersion increases with the number offlow cells in series (as depicted in FIG. 1B). Such dispersion resultsin a time lag in both the rise and fall of the sensorgram at thebeginning and end of sample introduction. These so-called “rise and falltimes” limit the ability to resolve fast reaction kinetics (i.e.,interactions with high rate constants). One way to solve this limitationis to increase the flow rate. Unfortunately, increasing the flow ratemeans increased sample consumption. There are also practical and designlimitations in terms of, for example, liquid pressure, which provide anupper limit for the flow rate.

In addition, temperature variations between flow cells can negativelyimpact sample analysis. Since refractive index, reaction kinetics andmass transport of the analyte to the sensing surface are all sensitiveto temperature, it is important that such measurements be carried out atcontrolled temperatures. Due to physical separation of the flow cells,and hence the sensing surfaces, temperature fluctuations between flowcells can be a source of measurement error. Further, the flow cellsdepicted in FIGS. 1A and 1B do not permit controlled sample delivery todiscrete areas within a single flow cell, nor do they allowimmobilization of different ligands to discrete sensing areas within asingle flow cell. Rather, such modifications are only achieved withinseparate flow cells, and thus are accompanied by the limitations asnoted above.

Accordingly, there is a need in the art for improved sample deliverytechniques within the context of flow cell-based detection instruments,such as the BIAcore instrument, as well as for other instruments ofsimilar design or operation. To that end, any instrument which detects ameasurable change in a property associated with a flow cell-basedsensing structure may benefit from improved sample delivery techniques.Such improvements should provide fast liquid exchange rates betweensample and buffer, maintain constant temperature control across multiplesensing areas, and permit a variety of sample delivery techniques tomultiple sensing areas within the flow cell.

The present invention fulfills these needs and provides further relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

In brief, the present invention is directed to the control of a fluidflow over a sensing surface within a flow cell and, more specifically,to the use of laminar flow techniques to position the fluid flow overone or more discrete sensing areas within the flow cell. Morespecifically, by varying the individual flow rates of at least twolaminar fluid flows, an interface between the two flows may be laterallymoved over the sensing surface within the flow cell. In this manner, theflows may be controllably positioned within the flow cell over one ormore discrete sensing areas, and further permits a wide range of surfacemodification and/or interaction at the discrete sensing areas.

One aspect of the invention provides a method of sensitizing a sensingsurface arranged to be passed by a liquid flow. The term “sensitizing”means any process or activation of the sensing surface that results inthe surface being capable of specifically interacting with a desiredanalyte. This method comprises providing a laminar flow of a first fluidand a laminar flow of a second fluid adjacent to the flow of the firstfluid so that the two laminar flows together pass over the sensingsurface with an interface to each other, with at least the first fluidbeing capable of sensitizing the sensing surface, and adjusting therelative flow rates of one or both of the two laminar flows to positionthe interface between the flows so that each laminar flow contacts adefined area of the sensing surface for selective sensitization thereof.

In one embodiment, the sensing surface is sensitized by contacting thesurface with a first fluid that sensitizes the same, and a second fluidthat does not sensitize the surface. In a variant of this embodiment,the procedure is repeated such that the first fluid is replaced by afluid that does not sensitize the sensing surface, and the second fluidis replaced by a fluid capable of sensitizing the sensing surfacedifferently than the first fluid to produce two differently sensitizedareas, optionally spaced apart by, or adjacent to, a non-sensitized areaof the sensing surface. In other embodiments, a stepwise gradient may beproduced by varying the relative flow rates of the laminar flows todisplace the interface laterally and provide a gradient-sensitized areaon the sensing surface or, alternatively, by continuously varying therelative flow rates of the laminar flows to generate a continuousgradient-sensitized area.

In still another embodiment, an additional laminar flow of a third fluidis provided on the other side of the flow of the first fluid so that thelaminar flow of the first fluid is sandwiched between the laminar flowsof the second and third fluids. This permits the flow of the first fluidto be positioned laterally on the sensing surface. If the second andthird fluids, which may be the same or different liquids, are notcapable of sensitizing the sensing surface, a streak or row ofsensitizing fluid may be controllably positioned on the sensing surface.By successively repeating the above procedure with at least onedifferent sensitizing first fluid, and with varied relative flow ratesof the second and third fluids, two or more rows of sensitized surfaceareas may be provided on the sensing surface.

In yet another embodiment, the method is used to produce either a row ormatrix of sensitized areas on the sensing surface. This may be achievedby repeating the procedure with a different sensitizing fluid or fluidsand applying the laminar flows at an angle, typically transversely, tothe original flow direction. Such rows or matrixes have a number ofbeneficial applications as described in more detail below.

Another aspect of the invention provides a method of analyzing a fluidsample for one or more analytes. This method comprises sensitizing asensing surface by immobilization of an analyte-specific ligand on thesensing surface by the methods described herein, contacting the sensingsurface with the fluid sample, and detecting interaction of analyte inthe fluid sample with the sensitized area or areas of the sensingsurface. One or more non-sensitized areas may be used as a referencearea or areas or, alternatively, one or more areas sensitized with acontrol ligand may be employed.

Still another aspect of the invention provides a method of analyzing afluid sample for an analyte where laminar flow of the fluid is toposition the sample flow on the desired sensitized area or areas. Themethod comprises providing a sensitized area on the sensing surface of aflow cell, the sensitized area being capable of selectively interactingwith the analyte; passing a first laminar flow of the fluid sample overthe sensing surface, but not in contact with the sensitized area;passing a second laminar flow of fluid that is not capable ofinteracting with the sensitized area over the sensing surface, thesecond laminar flow being adjacent to the first laminar flow and formingan interface therewith; adjusting the relative flow rates of the firstand/or second laminar flows such that the first laminar flow of fluidsample passes over the sensitized area; and detecting interaction ofanalyte in the fluid sample upon contact with the sensitized area.

Another aspect of the invention provides a method of analyzing a fluidsample for an analyte, where the whole sensing surface is sensitized andonly a part of the sensitized surface is contacted with the sample flow,whereas the other part is used as a reference area. In this way,bleeding as well as other uncontrollable events on the sensing surfacemay be referenced away. This method comprises providing a sensitizedsurface which is capable of selectively interacting with the analyte;passing a first laminar flow of the fluid sample over a first part ofthe sensitized surface (i.e., a first sensing area); simultaneouslypassing a second laminar flow of fluid that is not capable ofinteracting with the sensitized surface over the remainder of thesensitized surface (i.e., a second sensing area), wherein the secondlaminar flow is adjacent to the first laminar flow and the secondsensing area serves as a reference area; and detecting interaction ofanalyte in the fluid sample with the first sensing area. In a furtherembodiment, the relative flow rates of the first and/or second laminarflows are adjusted such that the first laminar flow passes over at leasta portion of the second sensing area, and interaction of analyte in thesample flow is detected with this newly contacted portion of the secondsensing area.

Yet other aspects of the invention provide methods of studying theassociation or dissociation of an analyte to or from a sensing surface,where laminar flow techniques are used to rapidly shift a sample fluidflow laterally in a flow cell to a position where the sample flowcontacts a sensitized sensing area. The method for studying associationcomprises providing a flow cell having a sensitized sensing area on asensing surface thereof which is capable of interacting with theanalyte; passing fluid sample in a first laminar flow through the flowcell; passing analyte-free fluid in a second laminar flow through theflow cell, the second laminar flow being adjacent to the first laminarflow and forming an interface therewith; setting the relative flow ratesof the fluid flows to place the interface between the laminar flows sothat the sample fluid flow does not contact the sensitized sensing area;changing the relative flow rates of the laminar flows to displace theinterface laterally so that the sample flow contacts the sensitizedsensing area; and determining association of analyte to the sensitizedsensing area. Similarly, the method for studying dissociation comprisesshifting the sample flow laterally so that the sample flow is no longerin contact with the sensitized sensing area; and determiningdissociation of analyte from the sensitized sensing area.

Still another aspect of the invention provides a sensor devicecomprising a flow cell having an inlet end and an outlet end; at leastone sensing surface on a wall surface within the flow cell locatedbetween the inlet and outlet ends; wherein the flow cell has at leasttwo inlet openings at the inlet end, and at least one outlet opening atthe outlet end, such that separate laminar fluid flows entering the flowcell through the respective inlet openings can flow side by side throughthe flow cell and contact the sensing surface. In one embodiment of thesensor device, the flow cell has two inlet openings and at least oneoutlet opening, and is of the Y flow cell type (i.e., having two inletsand a single outlet). In another embodiment, the flow cell has threeinlet openings and at least one outlet opening to establish threelaminar fluid flows in a sandwich fashion through the flow cell, and isof the Ψ flow cell type (i.e., having three inlets and a single outlet).

In still another embodiment, the flow cell is of the two-dimensionaltype. One variant of such a two-dimensional flow cell has at least twofirst inlets and at least one first outlet, and in an angularrelationship to the fluid pathway between them (usually transversely),at least two second inlets, and at least one second outlet. Arepresentative flow cell of this type is a two-dimensional Ψ cell.Another variant of a two-dimensional flow cell has the sensing surfacetumably mounted within the flow cell to permit it to be passed by fluidflows in two dimensions. The sensing surface may have at least twoadjacent sensing areas in the flow direction of the flow cell,particularly at least one sensing area and at least one reference area.Preferably, at least one sensing area is capable of specificallyinteracting with an analyte.

Yet another aspect of the invention provides a sensor system, comprisinga flow cell having an inlet end and an outlet end; at least one sensingarea on a sensing surface within the flow cell between the inlet andoutlet ends; the flow cell having at least two inlet openings at theinlet end, and at least one outlet opening at the outlet end; means forapplying laminar fluid flows through the inlet opening such that thelaminar fluid flows pass side by side through the flow cell over thesensing surface; means for varying the relative flow rates of thelaminar flows of fluids to vary the respective lateral extensions of thelaminar flows over the sensing surface containing the sensing area orareas; and, detection means for detecting interaction events at thesensing area or areas.

These and other aspects of this invention will be evident upon referenceto the attached drawings and the following detailed description.Furthermore, certain references have been cited herein for the purposeof clarity and completeness. Such references are each incorporatedherein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (prior art) depicts the channels and valves in an IFC as viewedfrom above, while the insert shows a side view of how the flow cells areformed by pressing the sensor chip against the IFC. FIG. 1B (prior art)illustrates sample flow through three of four flow cells (the empty andfilled circles represent open and closed valves, respectively).

FIGS. 2A and 2B illustrate a Y flow cell with two laminar flows (onebuffer flow and one sample flow).

FIG. 3A illustrates the sample interface positioned adjacent the sensingarea, while FIG. 3B shows the sample fluid covering the sensing area.

FIG. 4A illustrates a Y flow cell having two sensing areas with thesample flow initially adjacent to both sensing areas, while FIG. 4Bshows the interface displaced such that the sample flow contacts one ofthe sensing areas.

FIGS. 5A and 5B illustrate a representative Ψ flow cell having twobuffer flow inlets and one sample flow inlet.

FIG. 6 represents diffusion in the Y flow cell over contact time, t,where f is the flow rate, L is the length of the flow cell, and A is thecross section area of the flow cell.

FIG. 7A depicts the concentration of the sample at different contacttimes in a cross section of the Y flow cell, while FIG. 7B shows theconcentration of the sample at different contact times in a crosssection of the Ψ flow cell.

FIG. 8 illustrates formation of a gradient in the Y flow cell bychanging the sample and buffer flow rate during sensitization of thesensing surface.

FIG. 9 illustrates a Y flow cell having two discrete sensing areas.

FIG. 10 illustrates a Ψ flow cell having three discrete sensing areas.

FIG. 11A illustrates a representative two-dimensional (2D) Ψ flow cell,with FIG. 11B depicting a cross-section thereof.

FIGS. 12A through 12E illustrate generation of sensitized rows on thesensing surface, and an overlapping sensing area.

FIGS. 13A through 13E illustrate generation of a sensitized matrix ofthe present invention.

FIG. 14A illustrates a flow cell having two sensitized areas and onenon-sensitized area, with sample flow contacting all three areas. FIG.14B illustrates an alternative embodiment having two sensitized areasand one non-sensitized area, with sample flow contacting one sensitizedarea and the non-sensitized area.

FIG. 15 depicts a representative Y flow cell.

FIG. 16 illustrates a representative one-dimensional (ID) Ψ flow cell.

FIG. 17A illustrates the diffusion width of the 1D Ψ flow cell asmeasured at 2 mm and 10 mm from the inlet for different flow rates(i.e., different contact times), and FIG. 17B graphs the diffusion widthversus the contact time.

FIG. 18 graphs diffusion widths of different proteins and molecules atdifferent flow rates and at different concentration limits.

FIG. 19 is a schematic cross section of a Ψ cell with the relativesample concentration on the y-axis and length on the x-axis,perpendicular to the flow direction (the positioning width is the widthwhere the sample concentration is >99.9%, and the separation width iswhere the sample concentration is <0.1%).

FIG. 20A, 20B, 20C and 20D illustrate use of a Ψ flow cell for dialysis.

FIGS. 21A and 21B represent the part of the sensorgrams that show therise and fall times, respectively, for different flow cells, and FIG.21C shows a plot of rise time versus sample flow.

FIG. 22 presents a comparison of the liquid exchange rate constants forrepresentative Y, IFC3 and IFC4 flow cells.

FIGS. 23A through 23F illustrates sensitization at two discrete sensingareas, and selective analysis of analytes specific to such discretesensing areas.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, this invention is generally directed to the controlof a fluid flow over a sensing surface using laminar flow techniques tobring the fluid (also referred to herein as “sample flow” or “sample”)into contact with one or more discrete areas on the sensing surface(called “sensing areas”), as well as to the preparation of sensingsurfaces and in analytical methods, devices and systems related thereto.In the context of preparing a sensing surface, a ligand may beassociated with a discrete sensing area on the sensing surface (referredto herein as “sensitization”) by selectively contacting the discretesensing area of the sensing surface with a sample containing the ligandunder laminar flow conditions within a flow cell. The configuration anddimensions of the flow cells of this invention may vary widely dependingupon the specific application and/or detection method.

To this end, representative detection methods include, but are notlimited to, mass detection methods, such as piezoelectric, optical,thermo optical and surface acoustic wave (SAW) methods, andelectrochemical methods, such as potentiometric, conductometric,amperometric and capacitance methods. With regard to optical detectionmethods, representative methods include those that detect mass surfaceconcentration, such as reflection-optical methods, including bothinternal and external reflection methods, angle, wavelength or phaseresolved, for example ellipsometry and evanescent wave spectroscopy(EWS), the latter including surface plasmon resonance (SPR)spectroscopy, Brewster angle refractometry, critical anglerefractometry, frustrated total reflection (FTR), evanescent waveellipsometry, scattered total internal reflection (STIR), optical waveguide sensors, evanescent wave-based imaging, such as critical angleresolved imaging, Brewster angle resolved imaging, SPR angle resolvedimaging, and the like. Further, photometric methods based on, forexample, evanescent fluorescence (TIRF) and phosphorescence may also beemployed, as well as waveguide interferometers. While the presentinvention is hereinafter illustrated in the context of SPR spectroscopy,it should be understood that the invention is not limited in thismanner. Rather, any instrumentation or technique wherein a sample isbrought into contact with a sensing surface within a flow cell underlaminar flow conditions may benefit from this invention.

As mentioned above, the present invention involves contacting a samplewith one or more discrete sensing areas on a sensing surface underlaminar flow conditions within a flow cell. As used herein, the term“laminar flow” corresponds to a Reynolds number below 2000, andpreferably below 20. The Reynolds number (R_(e)) is used to describe thecharacter of a liquid flow over the sensing surface, and may beexpressed by the following Equation (1): $\begin{matrix}{R_{e} = \frac{{Vd}\quad\rho}{\mu}} & (1)\end{matrix}$where V is the average linear flow rate (m/s), d is the diameter of the“pipe” (m), p is the density of the fluid (kg/m³) and μ is the absoluteviscosity of the fluid (Ns/m²). In the context of a flow cell having arectangular cross section, the pipe diameter is more appropriatelyreplaced with the hydraulic diameter (D_(h)), which is given by fourtimes the cross sectional area divided by the perimeter of the flow cell(i.e., D_(h)=2wh/(w+h) where w and h are the width and height,respectively, of the flow cell). Thus, the Reynolds number of a flowcell having a rectangular cross section may more accurately berepresented by Equation (2): $\begin{matrix}{R_{e} = \frac{{VD}_{h}\rho}{\mu}} & (2)\end{matrix}$

It should be noted that Equation (2) assumes that the sensing surface isideally smooth and with a curvature that can be neglected. Thus, anyirregularities, pronounced curvature of the surface or sharp bendswithin the flow cell may lead to formation of local turbulence andshould be avoided. Further, laminar flow within the flow cell is bestachieved at some distance from entrance of the fluid into the flow cell.To this end, Weber and Prudy (Anal. Chim. Acta 100:531, 1978) haveproposed the following Equation (3) for ensuring laminar flow at adistance from a flow cell entrance (L_(e)):L_(e)≅0.05R_(e)D_(h)   (3)

As mentioned in the background section above, the rise and fall times ofa flow cell-based measurement (i.e., the time it takes for the sampleconcentration to rise from 0% to 100% over the sensing surface, and thenfall back to 0%) limits the ability to resolve fast reaction kinetics.At the beginning and end of sample introduction, the sample is dilutedby dispersion with the carrier solution (e.g., buffer). Thus, ratherthan instantaneous rise and fall times, there is some time lag due todispersion of the sample. Such dispersion can, as a first approximation,be described by a first order kinetic process according to Equation (4):$\begin{matrix}{{\frac{\mathbb{d}C}{\mathbb{d}t} = {k_{Lqx}\left( {C_{0} - C} \right)}}{or}{{\frac{\mathbb{d}C}{\mathbb{d}t} + {k_{Lqx}C}} = {k_{Lqx}C_{0}}}} & (4)\end{matrix}$where k_(Lqx) is the liquid exchange rate constant, C₀ is theconcentration of the sample and C is the concentration of the sample atthe sensing surface.

In the BIAcore instrument, the concentration of the sample at thesensing surface, C, is dominated by the dispersion in the flow cell.Multiplying Equation (4) with the integration factor e^(k) ^(Lqx) ^(t)and integrating the same gives the following Equation (5):C=C₀(1−e ^(−k) ^(Lqx) ^(t))   (5)This equation approximately describes the rise of the sampleconcentration at the sensing surface during a liquid exchange. The timerequired by the liquid exchange may, for example, be defined as the timeto reach 99% of the final value. Using Equation (5), rise time may thusbe expressed by Equation (6): $\begin{matrix}{0.99 = {\frac{C}{C_{0}} = {\left. \left( {1 - {\mathbb{e}}^{{- k_{Lqx}}t}} \right)\Rightarrow{{rise}\quad{time}} \right. = {t_{0.99} = \frac{4.6}{k_{{Lqx}\quad 0.99}}}}}} & (6)\end{matrix}$Thus, if the rise time to 99% is known, the liquid exchange rateconstant, k_(Lqx), can be calculated from Equation (6). Similarly, thefirst order equation for the liquid exchange during the fall isdescribed by Equation (7):C=C ₀ ·e ^(k) ^(Lqx) ^(t)   (7)which gives the following Equation (8) for the time it takes to fall to1% of the plateau value: $\begin{matrix}{0.01 = {\left. \left( {\mathbb{e}}^{k_{Lqx}t} \right)\Rightarrow{{fall}\quad{time}} \right. = {t_{0.01} = \frac{4.6}{k_{{Lqx}\quad 0.01}}}}} & (8)\end{matrix}$

To obtain an experimental relationship between rise time (or fall time)and flow rate, the rise time to 99% of the steady state concentration(or fall time to 0.01%) can be measured and plotted against sample flowaccording to Equation (9): $\begin{matrix}{{{Rise}\quad{time}} = \frac{V_{a} \cdot 60}{{Sample}\quad{flow}}} & (9)\end{matrix}$where V_(a) corresponds to the volume of sample (μl) that must bedisplaced during an exchange of fluid. In Equation (9), sample flow isexpressed as a sample volume per unit time (μl/min), and the rise timeis measured in seconds (hence the presence of the 60 sec/minconversion). Experimental results for representative flow cells of thisinvention are presented in Example 4. By determining V_(a) fromexperimental data, Equation (9) may be used to calculate rise times fordifferent sample flows.

Further, Equation (9) may also be used to obtain an expression for thetime to rise to 99% of the original concentration. Combining Equations(6) and (9), the liquid exchange rate constant may be expressed asEquation (10): $\begin{matrix}{K_{Lqz} = \frac{4.6}{\left( \frac{V_{a} \cdot 60}{{Sample}\quad{flow}} \right)}} & (10)\end{matrix}$Under appropriate conditions, this equation may be used to calculate theliquid exchange rate constant for different flow cells. The largerK_(Lqx), the faster the reaction kinetics that can be measured. Acomparison of liquid exchange rates is presented in Example 4 fordifferent flow rates through representative flow cells of thisinvention.

With regard to suitable flow cells for use in the practice of thisinvention, such flow cells may assume a number of forms, the design ofwhich may vary widely depending upon the intended application and/oruse. While several representative flow cells are disclosed herein forpurpose of illustration, it should be recognized that any type of flowcell which is capable of contacting a liquid sample to a sensing surfaceunder laminar flow conditions may be employed in the practice of thisinvention.

In one embodiment, a flow cell of this invention has two inlets and oneoutlet, such that two liquid streams enter the flow cell via theirrespective inlets and travel through the flow cell side-by-side andunder laminar flow conditions, exiting the outlet. A sensing surface islocated along a wall portion of the interior volume of the flow cellsuch that at least one of the liquid streams contacts the sensingsurface. A representative flow cell of this embodiment is depicted inFIGS. 2A and 2B, and is referred to herein as a “Y flow cell.”

Referring to FIG. 2A, a cross-sectional view of Y flow cell 200 isillustrated, having inlet arms 210 and 220 and outlet end 250. The flowcell has interior length 1, width w and height h (not shown). A firstfluid (such as a buffer), depicted by arrow 230, enters flow cell 200via inlet 210, and a second fluid (such as a sample), depicted by arrow240, enters via inlet 220 (in FIG. 2A, second fluid 240 is shaded forpurpose of illustration). The first and second fluids travel length l ofthe flow cell under laminar flow conditions such that interface 290separates first fluid 230 from second fluid 240, with both fluidsexiting outlet end 250 of the flow cell as depicted by arrow 270.

An isometric view of a representative Y flow cell is illustrated in FIG.2B. In this figure, Y flow cell 201 has inlet arms 235 and 245, eachcontaining inlets 215 and 225, respectively, and having a common outlet255. A first fluid, represented by arrow 265 enters the flow cell viainlet 215, while a second fluid, represented by arrow 275 enters theflow cell via inlet 225. The two fluids travel in the direction ofoutlet 255 under laminar flow conditions such that interface 295separates first fluid 265 from second fluid 275, with both fluidsexiting outlet 255 as depicted by arrow 285.

As the two fluids travel through the Y flow cell illustrated in FIGS. 2Aand 2B, at least one of the fluids comes in contact with a discretesensing area along a wall portion within the interior volume of the flowcell, as depicted by sensing areas 260 and 261 in FIGS. 2A and 2B,respectively. The interaction between the fluid and the sensing area mayinvolve a variety of interactions, as discussed in greater detail below.Such interactions may be detected by sensing techniques known to thoseskilled in the art which probe the sensing area from the “backside”—that is, from the opposite side of the sensing area in contactwith the fluid. Alternatively, such interactions may be detected bysensing techniques which probe the sensing area from the “frontside”—that is, from the side of the sensing area in contact with thefluid. Such detecting may be done at the same time that the fluid is incontact with the sensing area or at some subsequent time, and may bedone while the sensing area is associated with the flow cell or separatetherefrom.

By employing at least two laminar flows, it is possible to guide thefluids within the flow cell in a controlled manner, thus bringing thefirst fluid (such as a sample) in selective contact with a sensing areawithin the flow cell. For example, FIG. 3A depicts flow cell 300 whichis similar to the flow cell 200 of FIG. 2A, but having sensing area 320located approximately along the center-line of the flow cell. In thisembodiment, movement or displacement of the interface may be used tobring the sample flow into contact with the sensing area. Morespecifically, a sample flow (represented by arrow 350) and buffer flow(represented by arrow 340) enter the flow cell, travel the length of theflow cell under laminar flow conditions, and exit the flow cell asrepresented by arrow 360. For purpose of illustration, sample flow 350is shaded. The flow rates of the sample and buffer flows are selectedsuch that interface 380 is at a position within the flow cell such thatthe sample flow is not in contact with sensing area 320. The sampleand/or buffer flow rates are then adjusted to displace interface 380 toposition interface 381 as shown in FIG. 3B, thus bringing sample flow350 into contact with sensing area 320. In this embodiment, the rise andfall times, as discussed above, are limited only by the movement of theinterface from a first position not in contact with the sensing area(see interface 380 of FIG. 3A), to a second position such that thesample flow is in contact with the sensing area (see interface 381 ofFIG. 3B). The volume of sample required to move the interface from thefirst to second positions is a fraction of the volume of the flow cellitself.

In another aspect of this embodiment, multiple sensing areas may beemployed within the flow cell. As illustrated in FIG. 4A, flow cell 400has sensing areas 420 and 430, with sample flow (the shaded fluiddepicted by arrow 455), buffer flow (depicted by arrow 465), andinterface 470 such that the sample flow is not in contact with either ofsensing areas 420 or 430. The flow rates of sample and buffer are thenadjusted to bring sample flow 455 into contact with sensing area 430 bymovement of interface 470 in FIG. 4A to a location between sensing areas420 and 430, as represented in FIG. 4B as interface 471. The advantagesof moving the interface in this manner are as discussed above inreference to FIGS. 3A and 3B. In addition, sensing area 420 in contactwith the buffer flow can be used for a variety of purposes as discussedbelow, including use as an internal reference.

In another embodiment of this invention, a flow cell is disclosed havingmore than two inlets. Representative of this embodiment is a flow cellhaving three inlets as shown in FIG. 5A, also referred to herein as a “Ψflow cell”. Referring to FIG. 5A, sample flow (depicted by arrow 535)enters Ψ flow cell 500 by middle inlet 531. A first flow (as depicted byarrow 525) enters Ψ flow cell 500 via inlet 521, and a second flow(depicted by arrow 545) enters via inlet 541. All three flows travelthrough the flow cell, side-by-side and under laminar flow, and exit theflow cell as represented by arrow 565. Again for purpose ofillustration, middle flow 535 and first flow 525 have been shaded. Thus,two interfaces 550 and 552 are present between the middle flow (e.g.,sample flow) and the first and second flows (e.g., buffer flows). Byadjusting the relative flow rates of the three flows, both the positionand width of the middle flow may be selectively controlled. For example,as illustrated in FIG. 5B, middle flow 535 may be displaced toward thelower side wall of the flow cell by appropriate control of the flowrates of first flow 525 and second flow 545. Thus, interfaces 550 and552 of FIG. 5A are shifted to locations 551 and 553, respectively, asshown in FIG. 5B.

At low linear flow rates, the flow through both the Y and Ψ flow cellsis laminar and there is no active mixing of the fluid streams. In thecontext of the Y flow cell, the two fluids pass through the flow cell ata common flow rate, and the position of the interface is determined bythe incoming flow rates. The following Equation (11) describes thesituation in a thin layer flow cell (i.e., a flow cell with arectangular cross-section and with w>>h: $\begin{matrix}{{Interface} = {w \cdot \frac{{First}\quad{Flow}}{{{First}\quad{Flow}} + {{Second}\quad{Flow}}}}} & (11)\end{matrix}$where w is the width of the flow cell and h is the height of the flowcell. Thus, by varying the first and second flow rates, the interfacemay be moved across the width of the flow cell.

With respect to the Ψ flow cell, location of the two interfaces underlaminar flow conditions (i.e., “Interface 1” and “Interface 2”) may becontrolled in the flow cell by varying the first and second flow rates(“First Buffer Flow” and “Second Buffer Flow”) and sample flow rate(“Sample Flow”) as approximated by Equations (12a) and (12b).$\begin{matrix}{{{Interface}\quad 1} = {w \cdot \frac{{{First}\quad{Buffer}\quad{Flow}} + {{Sample}\quad{Flow}}}{{{First}\quad{Buffer}\quad{Flow}} + {{Second}\quad{Buffer}\quad{Flow}} + {{Sample}\quad{Flow}}}}} & \left( {12a} \right) \\{{{Interface}\quad 2} = {w \cdot \frac{{Second}\quad{Buffer}\quad{Flow}}{{{First}\quad{Buffer}\quad{Flow}} + {{Second}\quad{Buffer}\quad{Flow}} + {{Sample}\quad{Flow}}}}} & \left( {12b} \right)\end{matrix}$where w is the flow cell width (and provided w>>h).

A more precise calculation of the position of the interface(s) requirescorrection of the above equations with the expression for the velocityprofile (Brody et al., Biophyscial Journal 71:3430-3441, 1996). Thevelocity profile is a parable with a velocity of zero close to the flowcell walls and maximum velocity in the middle of the flow cell.Directing a middle flow with two adjacent flows with equal flow ratesplaces the middle flow in the center of the flow cell (as illustrated inFIG. 5A). However, if one of the two adjacent flow has flow rate of, forexample, 5% of the total flow rate, this flow will actually occupy morethan 5% of the cross sectional area of the flow cell. This is becausethe linear flow rate close to the flow cell wall is slower than thelinear flow rate in the middle. The same volume flow rate requires abroader part of the flow cell close to the wall than at the middle ofthe flow cell.

In addition, directing a sample flow within a flow cell using a separateflow (e.g., a buffer) requires that the diffusion of the sample belimited to the region close to the interface between the two flows.Otherwise, diffusion will interfere with the directionality of thesample flow and, rather than two (or more) distinct flows, a “smear” ofsample and buffer will result. Since diffusion is a time-dependentprocess, the linear flow rates of the sample and buffer should beselected to limit diffusion in close proximity to the interface.Diffusion in a flow cell can be viewed as a one-dimensional phenomenonsince the concentration gradient parallel to the interface isnegligible. As long as the concentration at the side walls in the flowcell is constant, the width of the flow cell can be assumed to beinfinite. The contact time at the interface is the same as the time ittakes for the flow to transfer a molecule through the flow cell at acertain linear flow rate as illustrated in FIG. 6, where t is thecontact time (sec.), f is the total volume flow rate (m³/sec), A is thecross sectional area of the flow cell (m²), u is the linear flow rate(m/sec) and D is the diffusion constant (m²/sec). Thus, contact time canbe expressed as the length of the flow cell divided by the linear flowrate, and the average diffusion width is approximately √{square rootover (Dt)}. The dark area of FIG. 6 corresponds to 100%, the gray areacorresponds to 100-50%, and the light gray area corresponds to 50-0% ofthe original sample concentration.

The expression for concentration as a function of the distance and timeis derived from Fick's second law, which has the solution of Equation(13) for the one-dimensional diffusion encountered with a Y flow cell:$\begin{matrix}{{c\left( {x,t} \right)} = {\frac{C_{0}}{2}\left( {1 + {\frac{2}{\sqrt{\pi}}{\int_{0}^{\frac{x}{2\sqrt{Dt}}}{{\mathbb{e}}^{- \beta^{2}}\quad{\mathbb{d}\beta}}}}} \right)}} & (13)\end{matrix}$In short, Equation (13) is the expression for concentration as afunction of distance and time, where x (distance) is measuredperpendicular from the interface (i.e., x is 0 at the interface).Similarly, in the context of the Ψ flow cell, the expression for theconcentration as a function of distance and time is given by Equation(14): $\begin{matrix}{{c\left( {x,t} \right)} = {\frac{C_{0}}{2}\left( {1 + {\frac{2}{\sqrt{\pi}}{\int_{0}^{\frac{x}{2\sqrt{Dt}}}{{\mathbb{e}}^{- \beta^{2}}{\mathbb{d}\beta}}}}} \right)}} & (13)\end{matrix}$FIGS. 7A and 7B are a graphical representations of Equations (13) and(14), respectively, for different contact times, where the x-axis is thedistance x from the interface as discussed above, and the y-axis is therelative concentration (ranging from 0 to 100%).

In the preceding discussion, representative flow cells of this inventionare disclosed which are capable of controlling laminar flow within theflow cell, particularly with respect to sample flow position and width.Such flow cells have a variety of uses. In one embodiment, the flow cellmay be used to selectively contact a sample with a sensing area withinthe flow cell. In this manner, a flow cell having multiple sensing areasmay be utilized, with the sample flow being selectively contacted withone or more of the sensing areas by the techniques discussed above. Forexample, FIGS. 4A and 4B discussed above illustrate a flow cell havingtwo sensing areas, with sample flow being selectively contacted with oneof the sensing areas. However, it should be recognized that multiplesensing areas may be employed, and that the sample flow may beselectively contacted with any of the sensing areas under conditions oflaminar flow.

In another aspect, the sample flow contains a ligand that is used tosensitize a discrete sensing area within a flow cell. As used herein,“sensitize” or (“sensitization”) means any process or activation of thesensing area that results in the sensing area being capable ofspecifically interacting with a desired analyte. The resulting surfaceis referred to herein as a “sensitized sensing area” or a “sensitizedarea.”

As used herein, the terms “ligand” and “analyte” are to be construedbroadly, and encompass a wide variety of interactions. For example, thesensing area of the flow cell may be sensitized by immobilization of ananalyte-specific ligand thereto. Representative ligands in this contextinclude, but are not limited to, the following (in the following list, arepresentative binding partner is parenthetically identified): antigen(specific antibody), antibody (antigen), hormone (hormone receptor),hormone receptor (hormone), polynucleotide (complementarypolynucleotide), avidin (biotin), biotin (avidin), enzyme (enzymesubstrate or inhibitor), enzyme substrate or inhibitor (enzyme), lectins(specific carboxyhydrate), specific carboxyhydrate (lectins), lipid(lipid binding protein or membrane-associated protein), lipid bindingprotein or membrane-associated protein (lipid), polynucleotide(polynucleotide binding protein), and polynucleotide binding protein(polynucleotide), as well as more general types of interactions such asprotein (protein), protein (polynucleotide), polynucleotide (protein),DNA (DNA), DNA (RNA) and RNA (DNA) interactions, and interactionsbetween small organic synthetic compounds and proteins or nucleic acids.

Suitable ligands also include various chemical compounds that may beused, for example, to build a chemical library, including bifunctionalcompounds. One skilled in the art will recognize that ligands of thisinvention, and the corresponding analyte present in the sample flow,include a wide range of molecules that may be used to generate asensitized surface area to perform a variety of tasks including, but notlimited to, binding, hybridization, combinational chemistry, and othercomplex formations on the sensing surface. All such interactions areincluded within the scope of analyte-ligand interactions as this term isused in the context of this invention. Further, such interactionsinclude, but are not limited to, covalent as well as non-covalentforces, such as electrostatic, hydrophobicity, dispersion, van der Waalsor hydrogen binding forces, or any combination of the same.

As noted above, the location of the sample flow within the flow cell, aswell as the width of the sample flow, may be controlled in the practiceof the invention. This permits immobilization of a ligand in a narrowrow within the flow cell. For example, a gradient can be created withinthe flow cell by selectively directing sample flow over the sensingsurface during immobilization of the ligand thereon. This aspect of theinvention is illustrated in FIG. 8 wherein Y flow cell 800 containssensing areas 820, 830, 840 and 850. Sample flow (depicted by arrow 865)and buffer flow (depicted by arrow 875) are introduced into the flowcell, and flow therein under laminar flow conditions, exiting the flowcell as represented by arrow 885. Initially, sample flow is only incontact with sensing area 820, with interface 825 between sample flow865 and buffer flow 875 located between sensing areas 820 and 830. Thesample and buffer flows are then adjusted to bring the sample flow intocontact with sensing area 830, with interface 835 now located betweensensing areas 830 and 840. This is then repeated to bring interface 845between sensing areas 840 and 850, with sensing area 850 serving as acontrol. The length of time that the sample flows over sensing areas820, 830 and 840 yields a gradient with regard to the amount ofimmobilized ligand bound to the surface of each sensing area. It shouldbe noted that the various shades of gray of FIG. 8 are intended toillustrate the amount of bound ligand on each sensing area—that is, darkto light represents a higher amount of bound ligand to a lower amount ofbound ligand. While this aspect of the invention is illustrated in FIG.8 as a step gradient, a continuous gradient may similarly be generatedIn an alternative embodiment, a Y flow cell may be employed to sensitizediscrete sensing areas with different ligands. As illustrated in FIG. 9,sensing areas 910 and 911 may be sensitized with different ligands byintroducing a first sample flow containing a first ligand (depicted byarrow 920) into flow cell 900 under laminar flow conditions with asecond flow (depicted by arrow 930). In this manner, sensing area 911 issensitized with the first ligand. Second flow 930 may, in oneembodiment, be a buffer flow or, in another embodiment, be a secondsample flow containing a second ligand, in which case sensing area 910is sensitized with the second ligand simultaneously with sensitizationof sensing area 911 with the first ligand. When the second flow is abuffer flow, the buffer flow may be subsequently replaced with thesecond sample flow, in which case sensing area 910 is sensitized withthe second ligand subsequent in time to sensing area 911 beingsensitized with the first ligand. In this embodiment, the first sampleflow may remain, or be substituted with a first buffer flow.

In the context of a Ψ flow cell, further sensitization permeations arepossible. For example, two or more sensing areas may be sensitized withthe same or different ligands by selectively positioning the sample flowcontaining the ligand within the flow cell. Thus, in one embodiment,three sensing areas may be sensitized with the same or different ligandas illustrated in FIG. 10. Referring to FIG. 10, a sample flow (depictedby arrow 1010 and shaded for purpose of illustration) containing a firstligand is directed within flow cell 1000 between a first and a secondflow (depicted by arrows 1011 and 1012, respectively) such that sensingarea 1820 is sensitized with the first ligand. Sensing areas 1021 and1022 may then be sensitized by directing sample flow 1010 within theflow cell such that it contacts the desired sensing area, therebysensitizing the same with the ligand contained within the sample flow.In this manner, a number of sensing areas may be sensitized within theflow cell by any number of desired ligands.

In a further embodiment, a second sample flow may be passed over thesensing surface within the flow cell at some angle, typicallyperpendicularly to a first sample flow, thereby generating overlappingsensing areas. This embodiment is generally referred to herein as atwo-dimensional or “2D” flow cell. Use of such 2D flow cells in thepractice of this invention permits sensitization of overlapping sensingareas within a flow cell. A representative 2D flow cell of thisinvention is illustrated in FIG. 11A. This figure represents thecombination of two Ψ flow cells (a “2D Ψ flow cell”), such that thesample flows are at right angles to each other. While FIG. 11 A depictsa 2D Ψ flow cell, it should be understood that a 2D Y flow cell maysimilarly be employed, or a 2D Ψ flow cell having more inlets than thesix depicted in FIG. 11A (i.e., 3 inlets times Ψ T flow cells), or anycombination of Y and Ψ flow cells having any number of inlets.

Referring to FIG. 11A, flow cell 1100 has buffer inlets 1122, 1123, 1126and 1127, and sample inlets 1130 and 1132. A first sample flow (depictedby arrow 1131) passes through the flow cell and exists via outlet 1150.Similarly, a second sample flow (depicted by arrow 1133) can be appliedsequentially by passing through the flow cell at an angle relative tothe first sample flow, in this case perpendicularly, and exits viaoutlet 1152. Location and width of the first and second sample flows arecontrolled in the manner disclosed above with regard to the “Ψ-flowcell. Referring to FIG. 11B, a side view of FIG. 11A is presented. Theflow cell has a central plateau 1140 which directs the first and secondsample flows to contact sensing surface 1160. In the case of FIG. 11B,first sample flow 1131 is depicted.

By employing a flow cell of this invention, a wide variety of sensitizedsensing areas may be generated. For example, the 2D Ψ flow cell of FIG.11A may be used to make sensitized matrices as illustrated in FIG. 12.FIG. 12A shows a sample flow (depicted by arrow 1210) containing a firstligand being directed across sensing surface 1220 to yield sensitizedarea 1222, as shown in FIG. 12B. A second sample flow containing asecond ligand is directed across sensing surface 1220 as depicted byarrow 1230 of FIG. 12C, to yield sensitized area 1240, as represented inFIG. 12D, which overlaps with sensitized area 1222. This overlappingarea is thus sensitized with two different ligands applied sequentially.

It will be recognized that such selective sensitization of a sensingsurface permits a multitude of sensitization options. For example, tothe extent that the second ligand contained within second sample flow1230 of FIG. 12C interacts with the first ligand of sensitized area1222, the resulting overlapping area 1260 may be depicted as in FIG.12E. In short, a discrete area upon the sensing surface has beensensitized with a first ligand, followed by a second ligand. If thefirst ligand is, for example, a bifunctional reagent immobilized on thesensing surface, the second ligand may react with the immobilizedreagent to yield an area or “spot” on the sensing surface that has beenselectively modified with an immobilized bifunctional reagent (i.e., thefirst ligand), with a second ligand bond thereto.

Similarly, as illustrated in FIG. 13A, by directing different flows(depicted by arrows 1310, 1320 and 1330), each containing a differentligand, sensitized areas 1311, 1321 and 1331 of three differentimmobilized ligands are generated on sensing surface 1350 as shown inFIG. 13B. Referring to FIG. 13C, three sample flows depicted by arrows1360, 1370, and 1380, each contain the same or different ligands, aredirected across sensing surface 1350, yielding sensitized areas 1391,1392 and 1393. In this manner, each overlapping sensitized area of FIG.13D is analogous to the sensitized surface depicted in FIG. 12D, and mayfurther be represented as sensitized areas 1370 through 1378 as shown inFIG. 13E.

One skilled in the art will readily appreciate the wide range ofapplications that such a two-dimensional matrix affords. Basically, anyinteraction that may be captured at discrete locations on the sensingsurface of the flow cell may be measured. For example, a representativeinteraction is that of DNA sequencing by hybridization wherein thematrix may be prepared by the following coupling procedures usinglaminar fluid flows in two dimensions as disclosed above. First, thesensor surface (such as a dextran-coated surface having streptavidinbound thereto) has a ligand immobilized thereon (such as biotinylatedDNA oligonucleotides) in defined bands 1311, 1321 and 1331 asillustrated in FIG. 13B. Such bands are generated by passing sampleflows 1310, 1320 and 1330 over sensing surface 1350 as illustrated inFIG. 13A, with each sample flow containing a different biotinylatedoligonucleotide. Complementary DNA oligonucleotides are then directedacross the sensor surface as illustrated by arrows 1360, 1370 and 1380in FIG. 13C, yielding a pattern of immobilized complementary DNAoligonucleotide as shown in FIG. 13E, wherein each area (1370 through1378) represents a different immobilized complementary DNA.

The liquid flow containing the active reagent can be positioned to anywidth, from the whole width of the sensor area to very narrowdimensions. Different liquids and reaction conditions can be applied inthe liquid streams that position the reagents. Such situations can beused to protect active intermediates formed when neighboring lines areformed before the second dimension reagents are introduced. In fact,this invention may be used to perform any kind of chemical syntheses ondefined areas on a sensor surface. Organic solvents may be used wherereagents are difficult to dissolve in water. It is also possible to usethe diffusion of substances from organic solvents into water phases inthe flow system in order to protect, for example, proteins fromdenaturation in contact with organic liquids. Further, chemicallibraries can be produced by stepwise reactions on sensor surfaces.Complex molecules may be built with relatively few molecular buildingblocks. This invention may also be used for building polymers in definedareas on a sensor surface, such as peptides or oligonucleotides, indefined sequences. By using protection or deprotection in the differentdimensions, as well as larger or smaller areas for the activation,defined sequences can be built.

Another application of this invention is for studying how multiplebiomolecular complexes are formed and how they function. One example isepitope mapping of an antigen to find the binding sites for a series ofantibodies in relation to each other. By the procedure described fortwo-dimensional building of reaction areas, epitope mapping may thus beperformed. This can, for example, be done by directly measuring theinteractions for the formation of complexes by techniques such as SPRdetection, or analysis of bound material after the last molecule hasbeen introduced by fluorescence. An exemplary procedure is as follows:on a surface covered with RAMFc antibody, different lines are formed inone direction for all analyzed antibodies directly from culture fluid;the whole surface is covered by blocking antibody by one interaction byan irrelevant antibody; the whole surface is covered by antigen thatabsorbs to all lines with immobilized primary monoclonal antibody; inthe second dimension, the same antibodies are introduced and the formedcomplex for the second interaction for each area is measured; andregeneration of the whole surface is performed. Another situation of asimilar type is when a series of, for example, proteins form an activecomplex and the order of adsorbing substances is critical. Differentcombinations of substance introduction can be introduced and theresulting reaction pattern observed.

In a further aspect of this invention, sensing surfaces are disclosedhaving one or a multiple of sensitized sensing areas thereon. Suchsensing surfaces may be used for a wide variety of applications. Forexample, such surfaces may be sensitized with a different ligand at eachsensitized area. A sample may then be contacted with all the sensitizedareas and, based on the position of the sensitized area on the sensingsurface, interaction between the sample and any given ligand determined.Contacting the sensing surface with the sample in this embodiment neednot occur within a flow cell since the entire surface may be contactedwith the sample. Alternatively, if selective contacting of the sensingsurface with the sample is desired, such contacting may occur within aflow cell of this invention.

In another aspect of this invention, a sample flow is directed bylaminar flow techniques over a sensing surface having one or morediscrete sensing areas. As discussed above with regard to sensitizationwith a sample flow containing a ligand, a sample flow containing ananalyte (as opposed to a ligand) may be directed by the laminar flowtechniques of this invention across a sensitized surface (which can besensitized by the laminar flow technique of this invention or othertechniques) thereby allowing interaction with ligands on the sensingarea. The laminar flow techniques described herein achieve extremelyfast rise and fall times which make it possible to measure fast reactionkinetics in addition to standard binding analysis.

In this regard, and in one embodiment of this invention, movement of theinterface may be used to bring a sample flow containing an analyte intocontact with a sensing area. This may be illustrated by reference toFIG. 3A. Referring to that figure, Y-flow cell 300 has sensitizedsensing area 320, and sample flow (the shaded fluid depicted by arrow350) and buffer flow (depicted by arrow 340) are adjusted such thatinterface 380 is at a position within the flow cell such that the sampleflow is not in contact with the sensitized sensing area. The sample andbuffer flow rates are then adjusted to move the interface to position381 as shown in FIG. 3B, thus bringing sample flow 350 into contact withsensitized sensing area 320. In this embodiment, the rise and falltimes, as discussed in greater detail above, are limited only by themovement of the interface from a first position not in contact with thesensing area (see FIG. 3A), to a second position such that the sampleflow is in contact with the sensing area (see FIG. 3B). The volume ofsample required to move the interface from the first to second positionsis a fraction of the volume of the flow cell itself. Thus, instead ofshifting from buffer flow to sample flow with valves at some distancefrom the sensing area (e.g., a volume of about 0.5 μl for the BIAcoreinstrument), the interface can be moved with only a fraction of thevolume of the flow cell (e.g., 0.05 μl). Since the rise time isproportional to the volume that has to be displaced, a tenfold decreasein volume reduces the rise time by about 10 fold. Similar advantages areachieved with shorter fall times.

Such fast rise and fall times are of necessity when measuring fastreaction kinetics. For example, the techniques of this invention mayused to study association and dissociation. In one embodiment, ananalyte may be passed over a sensitized sensing area. The sample flowmay then be displaced from contact with the sensitized sensing area, andthe dissociation rate can be detected. Alternatively, a sample flow maybe rapidly displaced onto a sensitized sensing area, thereby allowingfor the detection and analysis of association kinetics.

In another embodiment of this invention, multiple sensing areas may beemployed within a single flow cell for purposes of analysis. In oneaspect, the flow cell may contain two sensing areas. As illustrated inFIG. 4A, flow cell 400 has sensing areas 420 and 430, with sample flowcontaining analyte (the shaded fluid depicted by arrow 455), buffer flow(depicted by arrow 465), and interface 470 such that the sample flow isnot in contact with either of the sensing areas. The flow rates ofbuffer and sample are then adjusted to bring sample flow 455 intocontact with sensing area 430 by movement of interface 470 to a locationbetween sensing areas 420 and 430, as depicted in FIG. 4B as interface471. The advantages of moving the interface in this manner are asdiscussed above. Further, because the multiple sensing areas are locatedin close proximity within the same flow cell, time lag and temperaturevariations between the two sensing areas are negligible, which increasesthe reliability and accuracy of sample analysis.

Moreover, the ability to control location of the sample flow within theflow cell, in combination with multiple sensing areas, permits a widerange of applications. For example, still referring to FIGS. 4A and 4B,sensing area 420 may be a non-sensitized or sensitized sensing area.Thus, sensing area 420 of FIGS. 4A and 4B may be used as anon-sensitized or blank control (i.e., with no surface immobilizedligand), or a sensitized control (i.e., with immobilized ligand boundthereto). A blank control can detect non-specific binding to the sensingarea (e.g., to the dextran matrix), while a sensitized control willprovide information about non-specific binding to both matrix andimmobilized ligand. In this manner, bulk effects and non-specificbinding can be “subtracted out”. Such subtraction is best achieved whenthe immobilization levels of the ligand are the same for the sensitizedsensing area and sensitized control. This is particularly important whenhigh immobilization levels are used (e.g., when the analyte is a smallmolecule). This aspect of the invention is further illustrated inExample 5.

In another embodiment of this invention, multiple sensing areas may beemployed in a variety of methods. For example, as illustrated by FIGS.14A and 14B, flow cell 1400 may have ligand immobilized on sensitizedsensing areas 1440 and 1460, and non-sensitized sensing area 1450. Theligand immobilization may be accomplished using techniques describedabove to direct the sample flow containing the ligand over the sensingsurface. However, one skilled in the art will recognize thatsensitization of the sensing areas, prior to probing with sample flowcontaining analyte, may be effectuated by methods other than the laminarflow techniques herein disclosed. For example, discrete sensitizedsensing areas may be made by currently known techniques, such as surfacemodification techniques using masking (e.g., photolithography). A sampleflow containing an analyte may then be directed into the flow cell asrepresented by arrow 1430, such that the sample flow contacts sensingareas 1440, 1450 and 1460. In this manner, non-sensitized sensing area1450 may serve as a control, and sensitized sensing areas 1440 and 1460may have the same or different ligands associated therewith. Thistechnique has particular advantage in analysis of fluids containingmultiple analytes, such as analysis of body fluids, which analytes couldbe analyzed simultaneously using this technique.

Alternatively, as illustrated in FIG. 14B, flow cell 1400 may haveligand immobilized on sensitized sensing areas 1450 and 1460, withsensing area 1440 serving as a non-sensitized control. Both a sampleflow, containing analyte, (depicted by arrow 1432 and shaded forpurposes of illustration) and a buffer flow (depicted by arrow 1434) maybe directed into the flow cell such that the sample and buffer flowinterface 1465 is between sensing areas 1460 and 1450. In this manner,sensitized sensing area 1450 serves as the principle analyses area, withsensing areas 1440 and 1460 both serving as controls (i.e., sensing area1440—sample flow, no immobilized ligand; sensing area 1460—buffer flow,with immobilized ligand). In this embodiment displacement of the sampleflow containing the analyte over multiple discrete sensing areas can beaccomplished. Therefore, independent analysis of analytes containedwithin a sample flow may be done nearly simultaneously by merelydisplacing the sample flow over the sensing area of interest. This couldalso be accomplished using multiple buffer and sample flows.

The above techniques also allow for the detection of ion exchange in theflow cell (see Example 3). More specifically, diffusion over theinterface can be used for membrane-free dialysis (e.g., small moleculeswill diffuse across the interface faster than large molecules). Byemploying a chromatography eluent (e.g., in-line or sample fractions) asthe sample flow, the salt content (e.g., ionic exchange chromatography)or organic solvent content (e.g., reverse phase chromatography) willvary. This variation permits direct detection by injection and in-situdialysis in the flow cell, thus avoiding dilution or pre-dialysis of thesample.

In a further embodiment, an analyte that is difficult to solve in awater phase may be passed through the flow cell in an organic sampleflow, allowed to diffuse into an adjacent water flow, and then used inone or more of the techniques set forth above.

The following examples are offered by way of illustration, notlimitation.

EXAMPLES Example 1 Representative Flow Cells

This example discloses representative flow cells, as well as the usethereof in the context of this invention. In particular, different Y andΨ flow cells are illustrated.

A representative Y flow cell uses the inlets to flow channels 1 and 2,and the outlet to flow channel 2 of a commercially available IFC 4 for aBIAcore 1000 system (BIACORE AB, Uppsala, Sweden). Referring to FIG. 15,sample flow enters Y flow cell 1500 from inlet 1510, and the bufferenters the Y flow cell from inlet 1520. Both sample and buffer exit theY flow cell via outlet 1530. Both sample and buffer flows are directedinto the flow cell by cutting an additional channel in the IFC 4 toallow both sample and buffer to be run through the flow cellsimultaneously. The volume of the Y flow cell is 180 nl (i.e., threetimes the volume of commercially available BIAcore flow cells).

A representative one dimensional (“ID”) Ψ flow cell is depicted in FIG.16, where sample flow enters T flow cell 1600 via inlet 1610, withbuffer flows entering by inlets 1670 and 1630, and all flows exitingoutlet 1640. The Ψ flow cell is made in PMMA, and employs Pharmacia 500pumps with 500 μl Hamilton syringes for delivery of fluid flow to theflow cell.

Example 2 Diffusion of Fluid Flows

This example summarizes experiments directed to diffusion of fluid flowsas they pass through the representative Ψ flow cell of Example 1. Asmentioned above in the context of FIG. 6, directing fluid flows within aflow cell under laminar flow requires diffusion of the sample to belimited to a region close to the interfere between the flows. If this isnot the case, diffusion will interface with the directionality of thesample flow and, rather than distinct flows, a “smear” of flows willresult.

In this experiment, diffusion width is determined after about 0.3seconds, which is the time it takes to transport a molecule through theflow cell of the BIAcore instrument at a flow rate of 10 μl/min. Thediffusion width (see FIG. 17A) was measured as the width of a colorchange of an indicator as measured by ocular inspection with a PanasonicWV-Ks 152 video camera and a microscope mounted to the work processor.The diffusion width for different contact times at the interface wasfitted with √{square root over (Dt·C)}, where C is a dimensionlessconstant fitted for each pH (see FIG. 17B). The experimental diffusionwidth was then estimated for different contact times from the fittedconstant C.

Table 1 shows the experimental results and the theoretically calculatedvalues for the diffusion of protons. The theoretical values arecalculated with Equation (14). The pH change required to obtain a colorshift was measured with a pH meter and ocular inspection. In theseexperiments, the required pH change was 0.4 pH units for phenol red (PR)and 0.6 pH units for bromophenol blue (BB). Since concentration changeperceived as a color change in a 100 ml container may not be the same asfor a thin layer flow cell, the theoretical diffusion width for BB iscalculated for three different concentration changes (i.e., pH shifts).The column “diffusion of indicator” in Table 1 describes the broadeningof the color change band due to the diffusion of indicator into the acidfluid. The errors in the PR and the BB part of Table 1 show anincreasing tendency as the pH difference between the two fluidsdecreases. This behavior is believed to be due to a flattening of theconcentration gradient as the concentration difference between the twofluids decreases. A steep concentration gradient makes a sharp colorchange, while a flat concentration gradient makes a diffuse colorchange. Consequently, an incorrect estimation of the required pH changefor a color shift will give increasing errors as the concentrationdifference between the two fluids decreases. TABLE 1 Diffusion ofProtons into the Indicators Phenol Red and Bromophenol Red Phenol Red2.8 mM (pKa = 7.9, D = 3.4 * 10⁻¹⁰) Experimental Theoretical Exp. Conc.Theo. Indicator width PH change width diffusion Error HCL pH C (μm)]change (%) (μm)-] (μm) Absolute Relative 0.086 4.6 249 0.4 22.5 243 16−10 −0.04 1.08 3.5 189 0.4 22.5 187 16 −14 −0.07 1.9 2.9 157 0.4 22.5127 16 14 0.09 1.04 (20% 2.8 157 0.4 22.5 144 12 1 0.01 Glycerol) 1.04(40% 2.2 123 0.4 22.5 100 8 15 0.12 Glycerol) Bromophenol Blue 1.4 mM(pKa = 4.1, D = 2.71 * 10⁻¹⁰) Experimental Theoretical Exp. Conc.Indicator width PH change Theo. diffusion Error HCL pH C (μm) change (%)width (μm) (μm) Absolute Relative 0.086 4.4 247 2 82 231 14 2 0.01 1 50242 14 −9 −0.04 0.6 33 257 14 −24 −0.10 1.08 3.4 191 2 82 171 14 6 0.031 50 186 14 −9 −0.05 0.6 33 199 14 −22 −0.12 1.9 2 112 2 82 105 14 −7−0.06 1 50 126 14 −28 −0.25 0.6 33 144 14 −46 −0.41

In Table 1 above, C is a constant from the fit of √{square root over(Dt·C)} to the experimental measured diffusion widths, “Exp. width” isthe diffusion width of protons into the indicator fluid obtained fromthe experimental data at the time 0.3 sec. (corresponding to the time ittakes to transport a molecule through a BIAcore™ flow cell at a flowrate of 10 μl/min) and “pH change” in the theoretical part is therequired pH change during an ocular inspection of the indicator fluid.In the BB part the theoretical diffusion width is calculated for threedifferent pH changes because the color change of the BB was difficult toestimate. The column “conc. change” is the corresponding concentrationchange to the estimated pH change (i.e., how many indicator moleculesthat have to be proteolysed before a color change could be seen), and“theo width” is the theoretical diffusion width calculated from therequired concentration change for a color change of the indicator. Thediffusion of the indicator into the acid fluid is calculated in thecolumn “Indicator diffusion.”

The pH for PR was adjusted 1.0 pH units below pKa for PR, while the pHfor BB was adjusted 0.1 pH units below the pKa. Therefore, the requiredconcentration change for a color change is larger for BB than for PR.The difference in adjusting the pH for the indicators arises from thedifficulties to distinguish between the blue, blue-green-yellow and theyellow color for BB. The estimation that 0.6 pH units is enough for acolor change of BB may not be accurate, and more probable is that therequired concentration change was between 1 and 2 pH units. The resultsin Table 1 show that the derived theory may be used to predict thediffusion in the thin layer flow cell. Further, the tests with phenolred show that it is possible to calculate the diffusion in more viscosemedia like glycerol with desirable precision.

To this end, FIG. 18 shows the calculated diffusion width for someproteins of various size, as well as some small molecules, while Table 2gives the calculated diffusion width in numbers. Further, Table 2provides the information that is needed to calculate how many narrowbands of immobilized molecules that can be placed on the sensing surfacewithin the flow cell. Looking at IgG, for example, at a flow rate of 100μl/min and a separation of 0.1%, the diffusion width between theinterface and 0.1% is 5 μm and the width between the interface and 99.9%is also 5 μm. If 99.9% of the original concentration is needed fordetection in a band with a width of 10 μm, then the separation width is30 μm (see FIG. 19). 30 μm narrow bands in flow cells having a width of500 μm gives 16 possible bands in the flow cell. The same calculationfor sucrose gives 10 bands in the flow cell. It should be noted,however, that the number of bands can be increased be employing ashorter sensing area, which will result in a decrease in the diffusionwidth, and thus an increase in the number of potential bands within theflow cell. TABLE 2 Diffusion Width (μm) for Some Proteins and SmallMolecules Flow 100 Protein/ D Flow 10 (μl/min) (μl/min) Molecule Mw([m²/s)] 0.1% 1% 10% 0.1% 1% 10% Urease 490,000 3.40E−11 14 11 6 5 3 2Immuno- 156,000 4.00E−11 15 12 6 5 4 2 globulin (IgG) Serum 64,0006.15E−11 19 15 8 6 5 3 albumin Bovine 12,000 1.50E−10 30 23 13 10 7 4insulin Sucrose 337 5.20E−10 56 42 24 18 13 8 Na⁺ 11 1.33E−09 90 68 3828 21 12 H⁺ 1 9.31E−09 240 180 100 75 57 31

Example 3 Ion Exchange/On-Line Dialysis

The FIG. 20 shows how an ion exchange or an on-line dialysis may beperformed employing the techniques of this invention. FIGS. 20A and 20Billustrate ion exchange with low ion concentration in the adjacentbuffer. FIGS. 20C and 20D demonstrate ion exchange when the ionconcentration in the adjacent buffer is high. The panels shown in FIG.20 are for two different flow rates: 10 μl/min in FIGS. 20A and 20C, and100 μl/min in FIGS. 20B and 20D. The distance between the two interfacesis 12 μm in 20A, 20B, 20C and 20D. Referring to FIG. 20A, the flow rateis 10 μl/min and the concentration of salt and protons for the adjacentbuffer is low. The salt concentration in the sample flow decreases to10% and the proton concentration decreases to 6% of the originalconcentration. In FIG. 20B the flow rate is 100 μl/min, the saltconcentration in the sample flow decreases to approximately 30% of theoriginal concentration, and the proton concentration decrease toapproximately 20% of the original concentration in the sample. In FIGS.20C and 20D the adjacent buffer has a high concentration of salt andprotons.

Example 4 Exchange Rates for Representative Flow Cells

A sucrose solution (5% by weight) was used to measure the liquidexchange rate in the representative Y flow cell of Example 1 (see FIG.15), and compared with a flow cell of a commercially available BIAcore2000 (referred to as an “IFC 3 flow cell”) and from a commerciallyavailable BIAcore 1000 (referred to as an “IFC 4 flowcell”). The IFC 3and IFC 4 flow cells are both 60 nl in volume, but different withrespect to the channels that lead to the flow cells and to placement ofthe valves. In this experiment, reference to the IFC 3 and IFC 4 flowcells include the channels, valves and 60 nl flow cells.

The rise time was measured as the time it takes to reach 99% of theplateau value for different flow rates through the tested flow cells.Plotting the rise time against the flow rate, and fitting this curvewith Equation (9) gives a constant V_(a). With this constant it ispossible to calculate both the liquid exchange rate for different flowrates and the liquid exchange rate constant for the flow cell.

A fast liquid exchange during the rise in the Y flow cell wasaccomplished as follows. Before the sample was introduced into the flowcell only buffer was running through the flow cell. The sample flowvalves shifted and the sample entered the flow cell. The sample flowfilled up only a narrow part of the flow cell. At the liquid exchange,the valve to the buffer flow closed and the sample flow displaced thebuffer and filled up the flow cell. The fall was done in the same way asthe rise. The buffer entered the flow cell and filled up only a narrowpart of the flow cell. The sample flow valve closed and the buffer flowdisplaced the sample fluid. The time it took to cover up the sensingarea corresponded to the movement of the interface over the sensing areaand the dispersion of the interface. In this context, the sensing areawas an approximately 1.6 mm by 0.17 mm area located between inlet 2 andoutlet 2 of FIG. 15 (with the total sensing surface being roughly 2.4 mmby 0.8 mm).

FIGS. 21A and 21B compare the sensorgrams for the Y, IFC 3 and IFC 4flow cells. To obtain an experimental relation between the rise time andthe flow rate the rise time to 99% of the steady state concentration wasmeasured and plotted versus the sample flow (see FIG. 21C) according toEquation (9).

Equation (10) was used to calculate the liquid exchange rate constantfor different flow rates. To get a valid estimate of the reaction rates,K_(Lqx)>>than the on-rate and the off-rate. The larger K_(Lqx) thefaster kinetics can be measured. The results of this calculation arepresent in FIG. 22, which shows a comparison of the liquid exchangerates for the different flow cells at different flow rates.

In the BIAcore IFC 3 and IFC 4 flow cells the interface is rinsed outover the length of the sensing area and the length of the detection areais 10 times the width. In the Y flow cell the interface is displacedover the sensing area in a direction parallel to the width of the flowcell (i.e., transverse to the side wall). Thus, the distance that theinterface must travel is 10 times less than for the IFC 3 or IFC 4 flowcells. This movement transverse to the flow direction, combined with thesmall dispersion in the Y flow cell, explains why the exchange of fluidscan be done much faster in the Y flow cell.

The faster liquid exchange during the rise for the Y flow cell than forthe fall (see FIG. 22) is due to the fact that the required sample flowis lower before the rise than during the fall relative to the total flowin the flow cell. The higher flow before the fall for the Y flow cell isrequired to cover the sensing area (i.e., the interface is further awayfrom the sample flow inlet). The buffer flow valve in the IFC 3 and IFC4 flow cells is placed closer to the flow cell than the sample flowvalves (i.e., the dispersion in the IFC3 and 4 flow cells is less forthe liquid exchange to buffer flow than for the liquid exchange tosample flow). These two effects together explain the improvement of therise compared to the fall.

Example 5 Sensitization and Analysis

This experiment illustrates the use of a representative Y flow cell ofthis invention to immobilize two different ligands on discrete sensingareas. Immobilization was done with two fluid flows passing through theflow cell side-by-side under laminar flow conditions. A BIACore 2000 wasemployed for this experiment, using the Y flow cell of FIG. 15. FIG. 23Ashows the result of the immobilization of a first ligand (i.e.,biotinylated oligonucleotide 15-mer, called “RI”) over sensing area 1and the outline of the Y flow cell during the immobilization. During theimmobilization there were no responses from sensing areas 2 and 3. Thesensorgram shows a bulk effect from the immobilization flow, but notfrom the buffer flow over sensing area 2 and 3. During theimmobilization of a second ligand (i.e., a different biotinylatedoligonucleotide 15-mer called “R2”) over sensing area 3, there was noresponse from sensing areas 1 and 2, as shown by FIG. 23B. Thesesensorgrams clearly show that the Y flow cell is very good forimmobilization of two different ligands in a single flow cell.

FIG. 23C shows the injection of an analyte (i.e., a oligo 16-mer, called“R4”, complementary to R1). The entire sensing surface was contactedwith the analyte. Even though the analyte was in contact with both ofthe sensitized areas (i.e., sensing areas 1 and 3), only the specificinteraction with sensing area 1 gave a response. The non-interactingligand and the non-sensitized area can be used as references. FIG. 23Cshows a bulk effect from all the sensing areas, this bulk effect issubtracted out in FIG. 23E. FIG. 23D shows the injection of a differentanalyte (an oligo 16-mer complementary, called “R5”, complementary toR2). The bulk effect is around 100 RU, but the bulk effect may besubtracted as shown in FIG. 23F.

From the foregoing, it will be evident that, although specificembodiments of the invention have been discussed herein for purpose ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1.-44. (canceled)
 45. A method of sensitizing a sensing surface arranged to be passed by a liquid flow within a flow cell, comprising: providing a laminar flow of a first sensitizing fluid capable of sensitizing the sensing surface, and a laminar flow of a non-sensitizing fluid adjacent to the flow of the first sensitizing fluid such that the two laminar fluids flow together over the sensing surface with an interface to each other, adjusting the relative flow rates of the first sensitizing fluid and non-sensitizing fluid to position the interface such that the first sensitizing fluid contacts a first discrete sensing area of the sensing surface for selective sensitization thereof, replacing the first sensitizing fluid by non-sensitizing fluid, and replacing the non-sensitizing fluid by the first sensitizing fluid or by a second sensitizing fluid that is capable of sensitizing the sensing surface, and adjusting the relative flow rates of the sensitizing fluid and the non-sensitizing fluid to displace the interface laterally such that the sensitizing fluid contacts a second discrete sensing area spaced apart from the first discrete sensing area of the sensing surface for selective sensitization thereof to produce two sensitized areas spaced apart by a non-sensitized area on the sensing surface.
 46. The method according to claim 45, wherein the second sensitizing fluid is capable of sensitizing the sensing surface differently than the first sensitizing fluid.
 47. The method according to claim 45, wherein sensitization of the sensing surface comprises immobilizing an analyte-specific ligand to the sensing surface.
 48. The method according to claim 45, wherein the analyte-specific ligand is selected from the group consisting of antigen, antibody, antibody fragment, oligonucleotide, carbohydrate, oligosaccharide, receptor, receptor fragment, phospholipid, protein, hormone, avidin, biotin, enzyme, enzyme substrate, enzyme inhibitor and organic synthetic compound.
 49. The method according to claim 45, wherein the flow cell has two inlet openings and at least one outlet opening.
 50. A method of sensitizing a sensing surface arranged to be passed by a liquid flow within a flow cell, comprising: providing a laminar flow of a first sensitizing fluid capable of sensitizing the sensing surface, and adjacent to the flow of the first sensitizing fluid on each side thereof a laminar flow of a non-sensitizing fluid such that the first sensitizing fluid flows sandwiched between two laminar flows of non-sensitizing fluid over the sensing surface with an interface to each non-sensitizing fluid flow, adjusting the relative flow rates of the first sensitizing fluid and the non-sensitizing fluids to position the interfaces such that the first sensitizing fluid contacts a first discrete sensing area of the sensing surface for selective sensitization thereof, replacing the first sensitizing fluid by a second sensitizing fluid capable of sensitizing the sensing surface, and adjusting the relative flow rates of the second sensitizing fluid and the non-sensitizing fluids to displace the flow of second sensitizing fluid laterally such that the second sensitizing fluid contacts a second discrete sensing area of the sensing surface for selective sensitization thereof.
 51. The method according to claim 50, wherein the second sensitizing fluid is capable of sensitizing the sensing surface differently than the first sensitizing fluid.
 52. The method according to claim 50, wherein in additional steps at least a third discrete sensing area is selectively sensitized.
 53. The method according to claim 50, wherein sensitization of the sensing surface comprises immobilizing an analyte-specific ligand to the sensing surface.
 54. The method according to claim 50, wherein the analyte-specific ligand is selected from the group consisting of antigen, antibody, antibody fragment, oligonucleotide, carbohydrate, oligosaccharide, receptor, receptor fragment, phospholipid, protein, hormone, avidin, biotin, enzyme, enzyme substrate, enzyme inhibitor and organic synthetic compound.
 55. The method according to claim 50, wherein the flow cell has three inlet openings and at least one outlet opening.
 56. A method of analyzing a fluid sample for at least one analyte, comprising sensitizing two discrete sensing areas on a sensing surface by the method according to claim 45, contacting the sensing surface with the fluid sample, and detecting interaction between the at least one analyte and the sensitized sensing areas, wherein a non-sensitized area on the sensing surface is used as a reference.
 57. A method of analyzing a fluid sample for at least one analyte, comprising sensitizing at least two discrete sensing areas on a sensing surface by the method according to claim 50, contacting the sensing surface with the fluid sample, and detecting interaction between the at least one analyte and the sensitized sensing areas.
 58. The method according to claim 57, wherein at least one non-sensitized area on the sensing surface is used as a reference.
 59. The method according to claim 57, wherein at least one sensitized area on the sensing surface is used as a reference.
 60. A method of analyzing a fluid sample for at least one analyte in a flow cell, comprising sensitizing two discrete sensing areas on a sensing surface by the method according to claim 45, and selectively contacting the fluid sample with one of the discrete sensing areas by passing the fluid sample through the flow cell under laminar conditions with a second fluid, wherein selective contact with the discrete sensing area is controlled by adjusting the relative flow rates of the fluid sample and the second fluid, and detecting interaction of the at least one analyte with the sensing area.
 61. The method according to claim 60, wherein the relative flow rates of the sample and the second fluid are adjusted to bring the sample fluid into contact with the sensitized sensing area that was not previously in contact with the sample fluid.
 62. The method according to claim 60, wherein a non-sensitized area on the sensing surface is used as a reference.
 63. The method according to claim 60, wherein a sensitized area on the sensing surface is used as a reference.
 64. A method of analyzing a fluid sample for at least one analyte in a flow cell, comprising sensitizing at least two discrete sensing areas on a sensing surface by the method according to claim 50, and selectively contacting the fluid sample with at least one of the discrete sensing areas by passing the fluid sample through the flow cell under laminar conditions sandwiched between second and third fluids, wherein selective contact with the at least one discrete sensing area is controlled by adjusting the relative flow rates of the fluid sample and the second and third fluids, and detecting interaction of the at least one analyte with the sensing area.
 65. The method according to claim 64, wherein the relative flow rates of the sample fluid and the second and third fluids are adjusted to bring the sample fluid into contact with one of the at least two discrete the sensitized sensing areas that was not previously in contact with the sample fluid.
 66. The method according to claim 64, wherein a non-sensitized area on the sensing surface is used as a reference.
 67. The method according to claim 64, wherein a sensitized area on the sensing surface is used as a reference. 